Purpose: The aim of the study was to formulate a microparticulate vaccine formulation for metastatic breast cancer by using a murine metastatic breast cancer cell line 4T1 for transdermal administration through microneedles. Breast cancer (BC) is the most commonly diagnosed malignancy and is the second leading cause of cancer related death in American women. Currently there are no FDA approved vaccines for breast cancer. For this reason development of a therapeutic breast cancer vaccine is an area of research that needs urgent attention offering these women a better chance of a cancer free life. Many therapeutic vaccine strategies are under clinical trials for breast and other types of cancers. Most vaccines being studied today, such as the gene transfer based vaccines require live cultured cells, which is time consuming and difficult to establish in many cancers. It is well known that breast cancer cells do not grow easily in vitro, significantly limiting the number of patients eligible for such clinical trials and ultimately vaccine therapy. A recent clinical trial evaluating vaccine-based therapy concluded that one of the major problems with gene-based cancer vaccine therapy is the delay in vaccine production which significantly limits the access of patients to the trial and subsequent therapy. Another potential problem is that the delay in vaccine production and subsequent administration could also result in a delay in treatment and progression of tumor metastasis resulting in increasing tumor burden and worsening prognosis. Therefore an optimum cancer vaccine requires a rapid production time, ease of delivery, and has the ability to be customizable for individual patients. Our microparticle-based vaccine approach, addresses many of the problems associated with the current vaccine therapies including the high vaccine costs. We have developed a novel formulation using sustained release polymers encapsulating antigens in a biodegradable matrix containing immune potentiator adjuvants. In this study our purpose has been to formulate and evaluate a micro particulate therapeutic vaccine to provide a new line of therapy for metastatic breast cancer.
Methods: Vaccine microparticles were prepared by encapsulating 4T1 tumor associated antigens in cellulose polymer using spray dryer technology. In vitro characterization of microparticles was conducted. The potential of the formulations to induce an immunogenic reaction was assessed by nitric oxide assay. We measured the amount of nitrite released by dendritic cells (DC) in presence of nanoparticulate formulations. The cytotoxicity of blank and peptide loaded nanoparticles was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Expression of surface co-stimulatory molecules on dendritic cells treated with vaccine microparticles and other controls were determined by flow cytometer. To check the efficacy of the vaccine, vaccine microparticles were administered using microneedles in Balb/c murine breast cancer model. The different groups that were studied included the following: Group1: Naïve mice, Group 2: Transdermal (TD) blank particles, Group 3: TD microparticulate vaccine, Group 4: TD microparticulate vaccine + Cyclophosphamide. Group 5: TD microparticulate vaccine + MF59 + Alum; Group 6: TD microparticulate vaccine + MF59 + Alum + cyclophosphamide; Group 7: Vaccine Suspension + Alum + MF59 + Cyclophosphamide. Metastasis of 4T1 tumor cells to different organs was measured by the 6-thioguanine clonogenicity assay.
Results: The yield of the microparticulate vaccine, following spray drying was 80±5% w/w. The particle size was 1-4 µm and the zeta potential was -7±2 mV. The microparticulate vaccines demonstrated significantly stronger immunogenicity. For example, there was significantly higher amount of nitric oxide released in the supernatant, of cells exposed to vaccine microparticles compared to blank microparticles. Likewise, CD40, MHC II and CD80, MHC I expression were significantly higher in the vaccine microparticles group compared to blank microparticles and vaccine suspension group. The immunized animals showed significantly lower tumor growth compared to the naïve animals. Significant increase in the CD4+ T cell population was observed. Different organs (lymph node, lungs and liver) were investigated to to identify metastasized 4T1 cells. Most of the immunized animal groups were rescued from metastasis.
Conclusion: The novel vaccine formulation showed potential in both in vitro and in vivo characterization experiments as it were able to produce strong specific immune response. Also it was able to keep the tumor volume significantly lower in treatment groups in comparison to control groups. So, the whole cell lysate vaccine microparticles formulated by spray drying can potentially be an effective treatment for patients with metastatic tumor. This vaccine can futher be used for preclinical and clinical studies and can be potentially used for personalized treatment of metastatic breast cancer in humans.
Rikhav Gala– Research Scientist, University of New Mexico, Atlanta, Georgia
Nihal Mulla– Assistant Professor, Drake University
Cherilyn D'Souza– Mercer University, Atlanta, Georgia
Martin D'Souza– Professor, Mercer University, Atlanta, Georgia